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WO2002028175A2 - Sousris transgenique pour recombinaison ciblee a mediation par cre-er modifie - Google Patents

Sousris transgenique pour recombinaison ciblee a mediation par cre-er modifie Download PDF

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WO2002028175A2
WO2002028175A2 PCT/IB2001/002246 IB0102246W WO0228175A2 WO 2002028175 A2 WO2002028175 A2 WO 2002028175A2 IB 0102246 W IB0102246 W IB 0102246W WO 0228175 A2 WO0228175 A2 WO 0228175A2
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organism
cre
gene
rxr
recombinase
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PCT/IB2001/002246
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WO2002028175A3 (fr
WO2002028175A9 (fr
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Pierre Chambon
Daniel Metzger
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Association Pour Le Developpement De La Recherche En Genetique Moleculaire (Aderegem)
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Priority claimed from FR0012570A external-priority patent/FR2814642B1/fr
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Priority to EP01986247A priority Critical patent/EP1322152A2/fr
Publication of WO2002028175A2 publication Critical patent/WO2002028175A2/fr
Publication of WO2002028175A3 publication Critical patent/WO2002028175A3/fr
Publication of WO2002028175A9 publication Critical patent/WO2002028175A9/fr

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Definitions

  • the present invention relates to a metazoan organism, with the exception of humans, and in particular a mouse, characterized in that at least one cell of this organism comprises at least one fusion protein between a recombinase Cre and a modified nuclear estrogen receptor allowing it to respond to synthetic antiestrogens but not to natural estrogens, and one or more DNA sequences of interest belonging to the genome of said organism into which one or more sites of recognition of said recombinase protein are inserted.
  • the invention also covers the methods using said organism for the mutagenesis and the analysis of the biological function of the DNA sequence (s) of interest, in particular of the gene(s) of interest, such as RXR ⁇ .
  • mice embryonic stem cells
  • ES cells embryonic stem cells
  • these techniques have proved not to be very informative in a large number of cases, in particular because the hereditary mutations thus generated were lethal during development and/or because their effects were pleiotropic.
  • strategies for conditional somatic mutagenesis have been developed, particularly in mice; they make it possible to selectively induce mutations in a given cell type (spatial control) or at a given time (temporal control) during the life of the animal.
  • a first strategy consists in combining the targeted homologous recombination with the site- specific recombination systems based on the use of recombinases which catalyze the recombination reaction between two short recognition DNA sequences. It has been shown that these site-specific recombination systems, although of microbial origin for the majority, could function in higher eukaryotes, such as plants, insects and mice (Sauer, 1994; Rajewsky et al . , 1996; Sauer, 1998) . Among the site-specific recombination systems commonly used, there may be mentioned the Cre/Lox (Sauer, 1998) and FLP/FRT (Kilby et al . , 1993) systems.
  • the strategy normally used consists in inserting the loxP (or FRT) sites into the chromosomes of ES cells by homologous recombination, or by conventional transgenesis, and then in delivering Cre (or FLP) for the latter to catalyze the recombination reaction.
  • the recombination between the two loxP (or FRT) sites may be obtained in ES cells (Gu et al . , 1993) or in fertilized eggs (Araki et al . , 1995) by transient expression of Cre or using a Cre transgenic mouse (Lakso et al . , 1992; Orban et al . r 1992).
  • somatic mutagenesis allows a spatial control of the recombination, because the expression of the recombinase is controlled by a promoter specific for a given tissue or for a given cell.
  • this strategy also has limitations because some somatic alterations can lead to a lethal phenotype at an early stage of development, thus preventing any subsequent biological or physiological study.
  • an insufficiently specific expression of the recombinase can lead to recombination events in a non-desired cell type (Betz et al . , 1996) which, if they occur early during embryogenesis, can cause recombination of the DNA in the majority of the adult tissues, and thereby complicate the analysis of the mutant phenotype.
  • a second strategy has consisted in controlling the expression of recombinases over time so as to allow temporal control of somatic recombination.
  • the expression of the recombinases is controlled by inducible promoters (Kiihn et al . , 1995; Saint-Onge et al . , 1996), such as the interferon-inducible promoter, for example.
  • This system also has limitations because it does not make it possible to obtain spatial control of the recombination.
  • the inventors have solved the problems mentioned earlier, which had not been resolved up until now, by combining the selection of novel mutations in the ligand-binding domain of the human nuclear estrogen receptor, the selection of a suitable hinge region between the two domains of the chimeric recombinase, and the selection of promoters suitable for directing the expression of the chimeric recombinase in a given tissue.
  • the present invention therefore relates to a metazoan organism, with the exception of humans, characterized in that at least one cell of said organism comprises at least: (i) one fusion protein comprising sequentially:
  • a polypeptide comprising the ligand-binding domain of the human nuclear estrogen receptor, or of a vertebrate nuclear estrogen receptor, and their natural variants or one of their fragments, said polypeptide exhibiting at least one mutation relative to the wild-type form of said ligand-binding domains, or of their natural variants, or of their fragments, and said fusion protein having a negligible, or even zero, recombinase activity in the presence of a natural ligand, such as for example estradiol, and a recombinase activity induced by a small quantity of synthetic ligand endowed with antiestrogenic activity, such as for example Tarn and OHT; (ii) one or more gene or intergenic DNA sequences of interest naturally belonging to said genome of said organism into which one or more recognition sites of said recombinase protein are inserted, said DNA sequence (s) of interest being located in one or more of the chromosomes of the
  • metalazoan organism is understood to mean any animal organism, with the exception of humans, consisting of several cells. According to a preferred embodiment, it is a vertebrate such as for example a mammal, a bird, a fish. Preferably, it is a mammal such as for example a bovine, a porcine, a caprine, an ovine, an equine, a rodent. According to a more preferred embodiment, it is a rodent such as mice or rats.
  • recombinase protein is understood to designate recombinases of the family of integrases which catalyze the excision, insertion, inversion or translocation of DNA fragments at the level of specific sites of recognition said recombinases (Sternberg et al . , 1986, Sauer, et al . , 1990; Barbonis et al . , 1993; Kilby et al . , 1993; Sauer, 1994; Denisen et al . , 1995). These recombinases are active in animal cells (Sauer, 1994) .
  • the recombinase protein of the invention is preferably selected from the group of site-specific recombinases composed of the Cre recombinase of bacteriophage PI, the FLP recombinase of Saccharomyces cerevisiae, the R recombinase of Zygosaccharomyces rouxii pSRl, the A recombinase of Kl uyveromyces drosophilari ⁇ m pKDl, the A recombinase of Kl uyveromyces wal tii pKWl, the integrase ⁇ Int, the recombinase of the GIN recombination system of the Mu phage, of the bacterial ⁇ recombinase (Diaz et al . , 1999) or a variant thereof.
  • the Cre cyclization recombination recombinase which is a 38 KDa integrase of bacteriophage PI catalyzes, in the absence of cofactors, recombination between two DNA sequences of 34 basepairs called "loxP site" (Sauer et al . , 1990).
  • the position on one or more DNA molecules and the orientation of the loxP sites relative to each other determine the type of function of the Cre recombinase: excision, insertion, inversion or translocation.
  • the recombinase activity of Cre is an inversion when two loxP sites are inverted on the same DNA fragment, and an excision when the loxP sites are in the form of a direct repeat on the same DNA fragment.
  • the activity of the recombinase is an insertion when the loxP site is present on a DNA fragment, it being possible for a DNA molecule such as a plasmid containing a loxP site to be inserted at the level of said loxP site.
  • the Cre recombinase can also induce translocation between two chromosomes provided that a loxP site is present on each of them (Babinet, 1995) . More generally, the Cre recombinase is therefore capable of catalyzing recombination between one or more different DNA molecules provided that they carry loxP sites.
  • the FLP recombinase of the FLP/FRT system is a recombinase of 43 KDa from Saccharomyces cerevisiae which is capable of the same type of action as the Cre recombinase on DNA fragments containing FRT recognition sites (Kilby et al . , 1993).
  • the recombinase according to the invention is the Cre recombinase of bacteriophage PI and its natural or synthetic variants, and said sites of recognition specific for said Cre recombinase are preferably chosen from the group composed of the sequences Lox P, Lox 66, Lox 71, Lox 511, Lox 512, Lox 514.
  • variant of the recombinase protein is understood to mean all the wild-type recombinases or fragments thereof which may exist naturally and which correspond in particular to truncations, substitutions, deletions and/or additions of amino acid residues. These recombinases and fragments thereof are preferably derived from the genetic polymorphism in the population.
  • recombinase fragment is understood to mean any recombinase portion exhibiting at least one recombinase activity.
  • the expression variant of the recombinase protein is also understood to mean the synthetic variants for which the above modifications are not naturally present, but were introduced artificially, by genetic engineering for example.
  • the recombinases derived from chimeric fusion constitute synthetic variants according to the invention. Such recombinases have been described for example in Shaikh and Sadowski (2000) .
  • Said hinge region according to the invention comprises the D hinge region of the nuclear estrogen receptor, preferably the human nuclear estrogen receptor ⁇ , or one of its fragments.
  • the D hinge region (region 263 to 301 of the sequence SEQ ID No. 2) is a region situated between the
  • region 180-262 of the sequence SEQ ID No. 2 and the ligand-binding domain (region 302 to 552 of the sequence SEQ ID No. 2) .
  • this hinge region sequentially comprises at least (i) two amino acids corresponds to the introduction of a restriction site, of a "linker", or of an adapter, which are necessary for the cloning of the fusion gene, and (ii) one fragment of the D hinge region of the human nuclear estrogen receptor ⁇ , corresponding to amino acids 282 to 301 of the sequence SEQ ID No. 2.
  • said restriction site is an Xhol site and the two corresponding amino acids are leucine and glutamine.
  • the hinge region according to the invention has a size of at least 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 40, 45, 50, 55, 60, 65, 70, 100, 150, 200, 250, 292 amino acids. According to a preferred embodiment, said hinge region comprises at least 15 amino acids and at most 54 amino acids. More preferably still, the hinge region comprises 23 amino acids. The width of the hinge region influences the regulation of the recombinase activity by the ligand.
  • the hinge region according to the invention may consist of a peptide which is functionally equivalent to said D hinge region.
  • the estrogen receptors are proteins regulating the transcription of genes which mediate the action of estrogens in the target cells.
  • the ERs belong to the superfamily of nuclear receptors which have a common modular structure: (i) a variable N-terminal A/B region containing the constitutive transactivation activity AF-1, (ii) a DNA-binding central domain C (DBD) which is highly conserved between various species and allowing binding of the receptor to its specific DNA response element, (iii) and a ligand-binding domain (LBD), located in the C-terminal region of ER (for review articles and references see Evans, 1988; Beato et a 1 . , 1989; Gronemeyer, 1991; Green and Chambon, 1988; Parker, 1993; Simons, 1994) .
  • the nuclear estrogen receptors according to the invention are chosen from the human nuclear estrogen receptors, and from the nuclear estrogen receptors of vertebrates such as for example the various species of primates, bovines, porcines, ovines, caprines, felines, canines, equines, birds, fish, rodents, in particular rats and mice.
  • the source organism for the estrogen receptor according to the invention is characterized in that said ligand-binding domain (LBD) of the nuclear estrogen receptor, or its natural variants, or one of their fragments, is human and is chosen from the LBDs of the human nuclear ⁇ and ⁇ estrogen receptors (ER ⁇ and ER ⁇ ) .
  • LBD ligand-binding domain
  • this includes the LBD of the human nuclear estrogen receptor ⁇ corresponding to amino acids 302 to 552, or its natural variants, or one of their fragments.
  • the expression "natural variant” is understood to mean all the LBDs of the nuclear estrogen receptors or their fragments which may exist naturally, in particular in human beings, and corresponding in particular to truncations, substitutions, deletions and/or additions of amino acid residues. These natural variants are derived in general from the genetic polymorphism in the population, and have an activity which is not substantially modified compared with the wild-type receptor.
  • polypeptides homologous to the LBDs of the wild-type nuclear estrogen receptors, or to their variants, or to one of their fragments, and which exhibit certain modifications, in particular a deletion, addition, substitution of at least one amino acid, a truncation, an extension and/or a chimeric fusion.
  • nuclear receptor fragment is understood to mean any portion of the nuclear estrogen receptor LBDs exhibiting at least the LBD activity.
  • Said fusion protein according to the invention is therefore preferably Cre-ER and comprises the Cre recombinase protein to which is fused a portion of the D hinge region and the LBD (amino acids 282 to 595 of the sequence SEQ ID No. 2) of the mutated human nuclear estrogen receptor ⁇ (SEQ ID No. 2) .
  • the fusion protein according to the invention comprises at least the portion of the nuclear estrogen receptor having a ligand binding activity.
  • Said LBD of the nuclear receptor, or one of its fragments, has at least one mutation. This mutation is preferably chosen from the group: mutation (G521R) glycine to arginine at position 521 of the sequence SEQ ID No.
  • mutation is understood to mean any changes occurring in the sequence of the nuclear estrogen receptor, and in particular of the human nuclear estrogen receptor ⁇ , other than those present in its natural variants, and/or in its human or vertebrate homologues, and which substantially modify the activity of the recombinase protein fused to said receptor or to said ligand-binding domain, in response to the binding of a synthetic ligand endowed with antiestrogenic activity.
  • the mutations capable of being introduced into the LBD of the nuclear estrogen receptor there may be mentioned point mutations, deletions, insertions, substitutions.
  • the G521R mutation constitutes an LBD mutation of the ER according to the invention. This mutation is similar to the G525R mutation introduced into the mouse ER LBD (mER) which reduces the affinity for the natural ligand, estradiol, by about 1000 fold, without adversely affecting the binding of the synthetic ligand, 4-hydroxyTamoxifen (OHT) (Danielan et a 1 . , 1993) .
  • mER mouse ER LBD
  • OHT 4-hydroxyTamoxifen
  • Cre-ER T2 the fusion protein corresponding to the triple mutant G400V/M543A/L544A called Cre-ER T2 (Feil et al . , 1997).
  • This fusion protein exhibits a recombinase activity in cultured cells which is induced by the antiestrogen Tarn or OHT, but not by the natural ligand estradiol; moreover, the maximum activity of Cre-ER T2 is induced for Tarn or OHT doses less than those necessary to activate Cre-ER T .
  • Cre-ER T2 This increased sensitivity to Tarn or OHT of Cre-ER T2 compared with Cre-ER T has been verified in transgenic mice selectively expressing the chimeric recombinases in the basal layer of the epidermis, under the control of the cytokeratin 5 promoter (Indra et al . , 1999).
  • the inventors have observed that the translocation of Cre-ER T2 from the cytoplasm into the nucleus, as well as the excision of the DNA sequences flanked by loxP sites from a "reporter" gene are induced at doses of about ten times less than those necessary for Cre-ER T .
  • Cre-ER T3 With the aim of further increasing the sensitivity of the chimeric recombinase Cre-ER T2 to Tamoxifen, the inventors replaced the valine at position 400 in Cre-ER T2 with a glycine.
  • This novel fusion protein which corresponds to the double mutant M543A/L544A called Cre-ER T3 exhibits increased sensitivity to the synthetic antiestrogenic ligand such as Tarn and OHT, without the recombinase activity of this protein being induced by the natural ligand estradiol .
  • the inventors have thus shown that for 10 times lower injected Tarn doses, the recombinase activity in the cells of a transgenic Cre-ER T3 mouse is greater than that of a Cre-ER T2 mouse (see Example 5) .
  • the fusion protein according to the invention is Cre-ER T2 whose ER LBD exhibits the mutation G400V/M543A/L544A.
  • the fusion protein is Cre-ER , whose ER LBD exhibits the mutation M543A/L544A.
  • One of the objects of the present invention is therefore to provide a Cre-ER fusion protein which exhibits mutations in the human ER ⁇ LBD which are preferably chosen from the mutations G521R, G400V, M543A and L544A, whose recombinase activity is not induced by the natural ligands, and is highly induced by a small quantity of synthetic antiestrogenic ligand.
  • this fusion protein is Cre-ER T , Cre-ER T2 , Cre-ER T3 .
  • the present invention also relates to said fusion gene encoding said protein, said vector for expressing said protein, as well as the corresponding host cell, and the corresponding transgenic animal, which expresses said fusion protein in a particular cell type, preferably the epidermis, the liver or the adipose tissue.
  • the Cre-ER fusion protein of the present invention therefore comprises all or part of a nuclear estrogen receptor and a recombinase protein whose activity is inducible more strongly by the binding of said receptor or of said ligand-binding domain (LBD) of said receptor with a said antiestrogen than with a natural ligand.
  • LBD ligand-binding domain
  • Said Cre-ER fusion protein makes it possible to carry out a recombination between loxP sites, in a cell of the organism of the invention, following treatment with an antiestrogen. In the absence of treatment, or in the presence of concentrations of ligands such as the natural estrogens of up to 10 ⁇ M, no excision is observed. This system therefore makes it possible to release the recombinase activity of the chimeric protein at a given and chosen moment.
  • Said Cre-ER fusion protein may be expressed in cells containing loxP sites, without modifying the locus containing the loxP sites. The recombination at the level of the loxP sites takes place only after treatment with an antiestrogen such as Tarn or OHT. Furthermore, by expressing said Cre-ER fusion protein in an organism according to the invention, preferably an animal, under the control of a promoter with cellular specificity, it is possible to obtain recombination between loxP sites, specifically in these cells.
  • synthetic ligand is understood to mean any type of compound capable of binding to the nuclear estrogen receptor, and exhibiting agonist and/or antagonist activities, according to the species, the tissue or the cell type.
  • the synthetic ligand according to the invention is endowed with antiestrogenic activity, it is preferably the antiestrogenic therapeutic agent Tamoxifen (Tarn) , but also its metabolite 4-hydroxyTamoxifen (OHT) .
  • the antiestrogens ICI 164 384 and ICI 182 780 are also synthetic ligands according to the invention.
  • the present invention therefore provides a transgenic metazoan organism and more particularly a transgenic animal, and in particular a transgenic mouse: (i) in which at least one cell contains one or more chromosomal DNA sequences which are present in their natural chromatin context and are flanked (floxed) by loxP sites; (ii) which preferably expresses a chimeric Cre recombinase in a tissue-specific manner in one or more cell types of the organism (iii) whose chimeric Cre recombinase activity is negligible, or even zero, in the presence of estrogen; (iv) whose chimeric recombinase activity is activated by low concentrations of an antiestrogen (from 0.001 to 1 mg of Tamoxifen/mouse/day, for five days) ; (v) and finally whose Cre recombinase is capable of catalyzing, with an efficiency close to 100%, the site-specific targeted somatic recombination in the nucle
  • the doses of synthetic ligand injected into the metazoan organism according to the invention are low.
  • the term low is understood to mean quantities of less than or equal to 4 mg/adult mouse/day, preferably less than or equal to 2 mg/adult mouse/day, in a preferred manner less than or equal to 1 mg/adult mouse/day. According to an even more preferred mode, this quantity may be less than or equal to 0.5 mg, 0.25 mg, 0.10 mg, 0.075 mg, 0.05 mg, 0.025 mg, 0.001 mg per adult mouse and per day.
  • the efficiency of the targeted somatic recombination is estimated by techniques known to persons skilled in the art. This efficiency is estimated by the frequency of recombination events catalyzed by said recombinase. These events may be revealed by PCR or Southern Blotting; the recombination frequency being estimated by taking the ratio of the representation of the various alleles in the cells of a tissue. The frequencies of the various alleles may be estimated by assaying the intensity of the corresponding bands on an electrophoresis gel of a product of PCR amplification or of genomic DNA (Southern blotting) .
  • the use of the PCR makes this method of estimation extremely sensitive and makes it possible to detect the presence of cells of the organism whose genome has not undergone targeted site-specific recombination .
  • Another way of estimating the efficiency of the recombination may be carried out indirectly by immunohistochemistry, by analyzing the expression of the gene sequence to be inactivated for example.
  • said fusion protein is encoded by a fusion gene integrated into one or more of the chromosomes of said cell of said organism.
  • the fusion protein is encoded by a fusion gene integrated into an expression vector.
  • the fusion gene according to the invention is introduced into the cell in the form of an expression vector or of one of its fragments.
  • a "vector" is a replicon in which another polynucleotide segment (i.e. the fusion gene) is attached, so as to bring the replication and/or expression to the attached segment.
  • the vector may be in particular a bacterial plasmid DNA, a cosmid, a phage DNA, a viral DNA or a minichromosome (BAC, YAC and the like) .
  • a vector may be integrative, that is to say can integrate into the genome of the host cell or can exist in the form of an extrachromosomal replicon.
  • the expression vector is capable of replicating autonomously.
  • it is a fragment of an expression vector, preferably this fragment integrates into the cellular genome.
  • the expression vector or one of its fragments comprises at least the fusion gene and a promoter or expression elements which make it possible to direct and control the expression of said fusion protein in at least one cell of said organism.
  • the expression vector comprises, in addition, signals for initiation and termination of the transcription, as well as appropriate regions for regulation of the transcription. These various control signals are chosen according to the cellular host used.
  • the expression elements controlling expression is understood to mean all the DNA sequences involved in the regulation of the gene expression, that is to say the minimal promoter sequence, the upstream sequences, the activating sequences ("enhancers”), optionally the inhibitory sequences ("silencers”), the "insulator” sequences, and any other required sequence.
  • the fusion gene is placed under the control of tissue-specific or cell-specific or ubiquitous expression elements.
  • tissue-specific expression elements or tissue-specific promoter regions are chosen from the promoters which make it possible to obtain a specific, and preferably high, expression in one or more cells, tissues, cell types or organs of the organism according to the invention. These promoter regions may be heterologous or nonheterologous to the organism and may be naturally present or otherwise in the genome of the organism.
  • tissue-specific promoter regions there may be mentioned the promoter regions of the genes: - for cytokeratin, and more particular for cytokeratin 5 (K5) and cytokeratin 14 (K14), which directs the expression of the gene in the basal keratinocytes of the epidermis; for ⁇ -1-antitrypsin which directs the expression of the gene in the hepatocytes; for the adipocyte fatty acid binding protein 2 (aP2) which directs the expression of the gene in the adipocytes.
  • cytokeratin cytokeratin 5
  • K14 cytokeratin 14
  • ⁇ -1-antitrypsin which directs the expression of the gene in the hepatocytes
  • aP2 adipocyte fatty acid binding protein 2
  • said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER T .
  • said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER T2 .
  • said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER T3 .
  • said organism is characterized in that said promoter region is the cytokeratin 14 (K14) promoter region and said fusion gene Cre-ER T .
  • said organism is characterized in that said promoter region is the cytokeratin 14 (K14) promoter region and said fusion gene Cre-ER T2 .
  • said organism is characterized in that said promoter region is the cytokeratin 14 (K14) promoter region and said fusion gene Cre-ER .
  • said organism is characterized in that said promoter region is the ⁇ -1-antitrypsin promoter region and said fusion gene Cre-ER T . .
  • said organism is characterized in that said promoter region is the ⁇ -1-antitrypsin promoter region and said fusion gene Cre-ER T2 .
  • said organism is characterized in that said promoter region is the ⁇ -1-antitrypsin promoter region and said fusion gene Cre-ER T3 .
  • said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER T .
  • said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER T2 .
  • said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER T3 .
  • said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER T3 .
  • the organism according to the invention is characterized in that said fusion gene has the sequence SEQ ID No. 3 and encodes the Cre-ER T protein having the sequence SEQ ID No.
  • the organism according to the invention is characterized in that said fusion gene encodes, of sequence SEQ ID No. 5 the fusion protein Cre-ER T2 having the sequence SEQ ID No.
  • the organism according to the invention is characterized in that said fusion gene encodes, of sequence SEQ ID No. 7 the fusion protein Cre-ER T3 having the sequence SEQ ID No.
  • the tissue-specific promoter regions are more generally chosen from those which direct the expression of the fusion protein in a physiological system, an organ, a tissue, a cell type or a particular cell " , among which there may be nonexhaustively mentioned the nervous system in general, and in particular the brain, the cerebellum, the neurons, the motoneurons, the glial cells, the Schwann cells, the hypophysis, the hypothaia us, the pituitary gland, the hippocampus and the cortex, the heart, the ventricular cardiomyocytes and the auricular cardiomyocytes, the lungs, the bones, the eyes, and more particularly the retina and the crystalline lens, the skin and more particularly the dermis and the epidermis, the muscles, and more particularly the skeletal muscles, the cardiac muscle, the smooth muscles, the mammary gland, the gonads and more particularly the testes, the ovaries, the germ cells, the oocytes, the oogonias, the spermatozoa, the
  • the ubiquitous expression elements or ubiquitous promoter regions are chosen from the promoter regions which make it possible to obtain expression, preferably high expression, in all, or at least in a high proportion, of organs, or of tissues of the organism according to the invention. These promoter regions may be heterologous or nonheterologous to the organism according to the invention.
  • "so called" ubiquitous promoter regions there may be mentioned the cytomegalovirus (CMV) promoter (Schmidt et a 1 . , 1990) and the interferon-inducible promoter (Mxl) (Hug et al . , 1998; Arnheiter et al . , 1990) .
  • CMV cytomegalovirus
  • Mxl interferon-inducible promoter
  • the expression elements, or promoter regions according to the invention can ensure a constitutive or inducible control of the expression of the fusion gene.
  • the elements ensuring inducible expression there may be mentioned the eukaryotic promoter regions which are inducible by heavy metals (Mayo et al . , 1982; Brinster et aJ . , 1982; Seark et al . , 1985), by heat shock (Nover et al . , 1991), by hormones (Lee et al . , 1981; Hynes et al . , 1981; Klock et al . , 1987; Israel et al . , 1989), by interferon (Hug et al .
  • the inducible prokaryotic expression elements such as the E. coli Lac repressor system (LacR/operator/inducer) (Hu et al . , 1987; Brown et al . , 1987; Figge et al . , 1988; Deuschle et al . , 1990; Labow et al . , 1990), the E. coli tetracycline resistance system (Gossen et al . , 1992) (WO 94 04 672, EP 804 565) .
  • E. coli Lac repressor system LacR/operator/inducer
  • the fusion gene may be free of promoter regions or of expression elements and may be placed under the control of a promoter region or of endogenous expression elements.
  • the recombinant DNA technologies used for the construction of the expression vector according to the invention are those known and commonly used by persons skilled in the art. Standard techniques are used for cloning, isolation of DNA, amplification and purification; the enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases are carried out according to the manufacturer's recommendations. These techniques and others are generally carried out according to Sambrook et al .
  • the vector according to the invention or the vector fragments may be introduced into the host cell by standard methods such as for example microinj ection into a pronucleus, transfection by calcium phosphate precipitation, lipofection, elecfroporation, heat shock.
  • the fusion gene according to the invention preferably comprises in the 5' —» 3' direction: a DNA fragment encoding the Cre recombinase of bacteriophage PI or one of its variants; a DNA fragment of at least 45 nucleotides encoding at least either all or part of the D hinge region of a nuclear estrogen receptor, a region situated between the DNA-binding domain and the ligand- binding domain, or a peptide which is functionally equivalent to said D hinge region; and a DNA fragment encoding the ligand-binding domain (LBD) of a nuclear estrogen receptor or variants thereof, said fragment having at least one mutation conferring on LBD the capacity to respond to synthetic antiestrogens, but not to natural estrogenic agonists.
  • LBD ligand-binding domain
  • the fusion protein is directly introduced into the organism, or into a cell of the organism, it being possible for this introduction to be carried out by injection into a tissue or an organ in the case of an organism, or by microinjection in the case of a cell .
  • the DNA sequence of interest according to the invention is a gene or an intergenic sequence.
  • the DNA sequence of interest is a gene, it being possible for the function of the gene to be known or unknown.
  • the study of an organism according to the invention exhibiting modification of a gene or of any other genomic region of unknown function makes it possible to contribute to the definition of the function of this gene or of this intergenic region.
  • All the genes and intergenic regions of a metazoan organism are capable of being used in the context of the present invention; more particularly, there may be mentioned the RXR ⁇ , RXRp, RXR ⁇ , RAR ⁇ , RAR ⁇ , RAR ⁇ , SNF2p genes.
  • DNA sequence of interest naturally belonging to the genome of said organism, or DNA sequence of interest in its natural chromatin environment, is understood to mean an endogenous DNA sequence, such as an endogenous gene, present in the genome at its natural locus (loci) .
  • the organism according to the invention is an animal, in particular a mouse, characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER T having the sequence SEQ ID No. 4, or Cre-ER T2 having the sequence ID No. 6, or Cre-ER T3 having the sequence ID No. 8, said fusion gene being under the control of the cytokeratin K5 promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.
  • the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: - a fusion gene encoding the fusion protein Cre-ER T having the sequence SEQ ID No. 4, or Cre-ER T2 having the sequence ID No. 6, or Cre-ER T3 having the sequence ID No. 8, said fusion gene being under the control of the cytokeratin K14 promoter; - one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.
  • the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER T having the sequence SEQ ID No. 4, or Cre-ER T2 having the sequence ID No. 6, or Cre-ER T3 having the sequence ID No. 8, said fusion gene being under the control of the adipocyte fatty acid binding protein 2 (aP2) promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.
  • aP2 adipocyte fatty acid binding protein 2
  • the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER T having the sequence SEQ ID No. 4, or Cre-ER T2 having the sequence ID No. 6, or Cre-ER T3 having the sequence ID No. 8, said fusion gene being under the control of the ⁇ -1-antitrypsin promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.
  • the present invention also relates to methods of preparing a metazoan organism according to the invention.
  • a first method of preparation consists in the steps of: a) obtaining an embyronic stem (ES) cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence (s) of interest, located in one or more chromosomes, by homologous recombination; b) introducing said modified embryonic stem cell into an embryo of said organism; c) developing said embryo up to the stage of a fertile adult organism; d) crossing said fertile adult organism with a transgenic organism in which at least one of the cells expresses said fusion protein and obtaining the progeny derived from said crossing; and e) optionally, selecting, among said progeny, said metazoan organism.
  • ES embyronic stem
  • a second method of preparation consists in the steps of: a) obtaining a somatic cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence (s) of interest, located in one or more chromosomes, by homologous recombination; b) transferring the nucleus of said modified somatic cell into the cytoplasm of an enucleated recipient oocyte; c) developing the embryo obtained in step b) up to the stage of a fertile adult organism; d) crossing said fertile adult organism with a transgenic organism in which at least one of the cells expresses said fusion protein and obtaining the progeny derived from said crossing; and e) optionally, selecting, among said progeny, said metazoan organism.
  • the expression transfer of the nucleus or nuclear transfer is understood to mean the transfer of nucleus of a vertebrate live donor cell, of an adult organism or at the fetal stage, into the cytoplasm of an enucleated recipient cell of the same species or of a different species.
  • the transferred nucleus is reprogrammed to direct the development of the cloned embryos which may then be transferred into carrier females to produce the fetuses and the neonates, or used to produce cells of the internal cellular mass in culture.
  • a third method of preparation consists in the steps of: a) obtaining an embyronic stem (ES) cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence (s) of interest, located in one or more chromosomes, by homologous recombination; b) introducing said modified embryonic stem cell into an embryo of said organism; c) developing said embryo; and d) introducing said fusion protein into at least one cell of said embryo or of the organism obtained from the development of said embryo.
  • ES embyronic stem
  • a fourth method of preparation consists in the steps of: a) obtaining a somatic cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence (s) of interest, located in one or more chromosomes, by homologous recombination; b) transferring the nucleus of said modified somatic cell into the cytoplasm of an enucleated recipient oocyte; c) developing said embryo; and d) introducing said fusion protein into at least one cell of said embryo or of said organism obtained from the development of said embryo.
  • the insertion of the sites of recognition specific for the recombinase protein, in particular of the loxP site(s) for the Cre recombinase, into the DNA sequence of interest is preferably carried out by homologous recombination of the gene comprising said DNA fragment to be excised or inverted (two loxP sites) or respectively inserted or translocated (one loxP site) with a said modified gene comprising said DNA fragment to be excised flanked in 5' and/or 3' by said recombinase recognition site(s) according to the desired application, in particular the loxP sites.
  • the modified DNA fragment of interest may be integrated by homologous recombination into the genome of the cells of said organism before, at the same time, or after the step of introducing the fusion protein or of a transfer vector, or of a vector for expressing the fusion protein.
  • the DNA fragment of interest is introduced into pluripotent embryonic cells (ES cells) by the appropriate technique, such as for example elecfroporation, or the use of retroviral vectors, calcium phosphate precipitation, lipofection.
  • the DNA constructs intended for homologous recombination will comprise at least a portion of the DNA sequence of interest, in particular of the gene or of the intergenic sequence of interest into which will be introduced the desired genetic modification (s) , such as the introduction of at least one recombinase recognition site, and which will include regions of homology with the target locus.
  • desired genetic modification such as the introduction of at least one recombinase recognition site
  • positive and/or negative selectable markers for example the neo gene conferring resistance to the antibiotic G418, may be introduced.
  • the selectable marker used to make it possible to identify the homologous recombination events may be disruptive, and may be eliminated, if necessary, if it is itself flanked by recombinase recognition sites such as the loxP (or FRT) sites. This makes it possible to obtain mice in which the sole modification at the level of the modified locus is the insertion of recognition sites such as loxP.
  • the metazoan organisms obtained by the methods of preparation presented above can then be treated with a synthetic ligand endowed with antiestrogenic activity such as Tam and OHT.
  • the bringing of said cells of said organism into contact with said synthetic ligand is carried out by administration by the oral or topical route, or by injection and in particular, by intravenous, intramuscular, intraspinal, intracerebral, intraperitoneal injection.
  • the treatment with the synthetic ligand may be carried out by administration to the mother.
  • said synthetic ligand is preferably added to the culture medium, or injected into said cell.
  • This treatment or this bringing into contact makes it possible to inactivate or to modify a gene or an intergenic sequence of interest at a determined moment (temporal control) in a given tissue (spatial control) , and thus to make it possible to study the function of this gene or of this sequence at various periods during development or post- natally.
  • This is particularly advantageous for studying at the adult stage genes which are essential for the normal progress of embryonic development and whose inactivation is lethal in utero or perinatally.
  • Another object of the present invention is therefore to provide a method of conditional recombination, in particular excision, insertion, inversion, translocation, at the level of the DNA sequence of interest into which there is (are) inserted one or more sites of recognition for said recombinase protein, said DNA sequence of interest being located in one or more of the chromosomes of said genome of said cell of said organism according to the invention, characterized in that it comprises the steps of: (i) bringing at least one cell of said organism into contact with a synthetic ligand endowed with antiestrogenic activity; (ii) inducing the activity of the recombinase of said fusion protein by said synthetic ligand endowed with antiestrogenic activity.
  • the present invention therefore provides a method of conditional deletion of a DNA fragment in which a method of excision according to the invention is used and in which said DNA fragment (s) to be excised is (are) flanked by two recombinase protein recognition sites oriented as a direct repeat.
  • said DNA fragment (s) to be excised is (are) flanked by two recombinase protein recognition sites oriented as a direct repeat.
  • the present invention also provides a method of obtaining a metazoan organism, with the exception of humans, in which at least one cell possesses an allele of a gene of interest inactivated by a method of conditional deletion and in which the other allele of said gene of interest possesses a mutation, said method being characterized in that it comprises the steps of: a) obtaining a metazoan organism in which at least one cell of the germ line comprises said mutation in one of the alleles of said gene of interest; b) crossing said organism obtained in step a) with an organism according to the invention; c) selecting a progeny whose genome comprises a gene of interest in which one of the alleles possesses a mutation and the other allele possesses at least two recombinase protein recognition sites oriented as a direct repeat; and d) using the method of conditional deletion, according to the invention, of the DNA fragment of said allele of said gene of interest which
  • Such a method makes it possible to study and analyze the biological function of mutations other than deletions, and more particularly of the mutations observed in genes whose dysfunction causes a recessive genetic pathological condition.
  • This method is therefore particularly suited to the obtaining of transgenic animal models of recessively transmitted human genetic pathological conditions, the animal model being preferably a murine model.
  • the mutations are preferably point or limited mutations in exons or regulatory sequences such as insertions, deletions, substitutions.
  • the recombinase protein specific recognition sites are loxP sites and said recombinase protein is the Cre protein of the bacteriophage PI, or one of its variants.
  • the organisms capable of being obtained using the various methods above are also included in the scope of the invention. These organisms are preferably animals, and in a preferred manner rodents such as rats and mice, preferably mice.
  • the subject of the invention is a transgenic mouse K5-Cre-ER ⁇ /RXR ⁇ /L2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the basal keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse K5-Cre-ER ⁇ 2 /RXR ⁇ L2/L2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse K5-Cre-ER ⁇ 3 /RXR ⁇ L2/ 2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the basal keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse K14-Cre-ER ⁇ /RXR ⁇ L2/L2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse K14-Cre-ER ⁇ 2 /RXR ⁇ L2/L2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse K14-Cre-ER ⁇ 3 /RXR ⁇ 2/L2 whose RXR ⁇ gene may be selectively inactivated in the basal keratinocytes of the epidermis using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alopecia and/or hyperproliferation of the keratinocytes and/or an inflammatory reaction of the skin.
  • the subject of the invention is a transgenic mouse ⁇ AT-Cre-ER ⁇ /RXR ⁇ 2/ 2 whose RXR ⁇ gene may be selectively inactivated in the hepatocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse in particular alteration of the proliferation of the hepatocytes.
  • the subject of the invention is a transgenic mouse ⁇ AT-Cre-ER ⁇ 2 /RXR ⁇ L2/L2 whose RXR ⁇ gene may be selectively inactivated in the hepatocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse in particular alteration of the proliferation of the hepatocytes.
  • the subject of the invention is a transgenic mouse ⁇ AT-Cre-ER ⁇ 3 /RXR ⁇ L2/L2 whose RXR ⁇ gene may be selectively inactivated in the hepatocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse in particular alteration of the proliferation of the hepatocytes.
  • the subject of the invention is a transgenic mouse aP2-Cre-ER ⁇ /RXR ⁇ L /L2 whose RXR ⁇ gene may be selectively inactivated in the adipocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alteration of the metabolism of the lipids in the adipocytes and/or diabetes.
  • the subject of the invention is a transgenic mouse aP2-Cre-ER T2 /RXR ⁇ 2/L2 whose RXR ⁇ gene may be selectively inactivated in the adipocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alteration of the metabolism of the lipids in the adipocytes and/or diabetes .
  • the subject of the invention is a transgenic mouse aP2-Cre-ER T3 /RXR ⁇ 2/L2 whose RXR ⁇ gene may be selectively inactivated in the adipocytes using a conditional deletion method following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in said mouse alteration of the metabolism of the lipids in the adipocytes and/or diabetes .
  • said RXR ⁇ gene of said mouse is inactivated using a method according to the invention.
  • the present invention and in particular the metazoan organism and the cells derived therefrom are particularly useful for analyzing and studying the biological function of a DNA sequence of interest, whether it is a gene or an intergenic sequence in its natural chromatin environment. That is the reason why it is also within the scope of the present invention to provide a method of analyzing or studying the biological function of a DNA sequence of interest, in particular of a gene or an intergenic sequence, characterized in that it comprises the steps of: (i) bringing an organism according to the invention or cells isolated from said organism into contact with a synthetic ligand endowed with antiestrogenic activity; (ii) optionally inducing the expression of said fusion protein; (iii) revealing the recombination event catalyzed by the recombinase activity of said fusion protein; (iv) biochemical and/or physiological and/or phenotypic and/or behavioral study or analysis of said cell or of said organism.
  • the subject of the present invention is also the use of an organism according to the invention or of cells derived from said organism for carrying out a spatiotemporally controlled site-specific recombination of said DNA sequence of interest in its natural chromatin environment, with an efficiency of at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, in the presence of synthetic ligand in the cells of said organism expressing said fusion protein and with an efficiency at least less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or zero in the absence of synthetic ligand or in the presence of a natural ligand in the cells of said organism expressing said fusion protein.
  • said recombination is carried out in the epidermis, and more precisely in keratinocytes, in the adipocytes, in the malanocytes or in the hepatocytes.
  • the subject of the present invention is also its method of screening compounds capable of being used as a medicament for the preventive and/or curative treatment of pathological conditions associated with alteration of the expression (in the case, in particular, of an intergenic DNA sequence) and/or of the function (in the case of a gene DNA sequence) of said DNA sequence of interest, characterized in that it comprises the step of administering said compound to an organism according to the invention.
  • This organism may thus be used for the screening of compounds capable of constituting an active ingredient of a medicament intended for the treatment of pathological conditions associated with alteration of the expression- and/or of the function of said DNA sequence of interest.
  • the object of the present invention is to provide a method of screening compounds capable of being used as a medicament for the preventive and/or curative treatment of alopecia and/or of hyperproliferation of the keratinocytes and/or of inflammatory reactions of the skin, characterized in that it comprises the step of administering said compound to a mouse according to the invention.
  • the object of the present invention is also to provide a method of screening compounds capable of being used as a medicament for promoting, in particular, hepatic regeneration, characterized in that it comprises the step of administering said compound to a mouse according to the invention.
  • the object of the present invention is also to provide a method of screening compounds capable of being used as a medicament for the preventive and/or curative treatment of diabetes and/or for the treatment of the alteration of the metabolism of lipids, in particular of obesity, characterized in that it comprises the step of administering said compound to a mouse according to the invention.
  • the object of the present invention is also to provide a method of screening compound capable of being used as a medicament for the preventive and/or curative treatment of skin cancers, more specifically papillomas and melanomas at various stages of development, characterized in that it comprises the step of administering said compound to a mouse according to the invention.
  • Figure 1 Inactiva ion of the RXR ⁇ gene in the epidermis of " adult mice mediated by Cre-ER T and Cre-ER T2 , and induced by Tamoxifen.
  • Figure 1A Schematic representation of the wild-type genomic locus of RXR ⁇ (+) , of the floxed L2 RXR ⁇ allele, of the L ⁇ RXR ⁇ allele obtained after Cre-mediated excision of exon 4 (encoding the DNA-binding domain) , and of the RXR ⁇ (-)null allele (Kastner et al . , 1994).
  • the black boxes indicate the exons (E2 - E4) .
  • the enzymatic restriction sites and the position of the probe X4 are indicated.
  • the sizes of the BamHI fragments are indicated in kilobases (kb) : BamHI; C, Clal; E, EcoRI H, Hindlll; S, Spel; X, Xbal.
  • the arrow tips in the L2 and L " alleles indicate the Lox P sites.
  • FIG. IB The obtaining of RXR ⁇ ⁇ alleles mediated by K5-Cre-ER T induced by Tamoxifen is illustrated by Southern blot analysis of the DNA isolated from the epidermis, six weeks (lanes 1 to 3) or twelve weeks (lanes 4 to 6) after the first injection of Tamoxifen (1 mg) (AFT : "after first Tamoxifen treatment").
  • AFT "after first Tamoxifen treatment”
  • the genotype of the mice is as indicated, and the fragments digested with BamHI corresponding to the RXR ⁇ (+) , L2, L ⁇ , (-) alleles are described.
  • Figure 1C Tissue selectivity of the inactivation of RXR ⁇ mediated by Cre-ER T .
  • the wild-type alleles (+) , L2, L ⁇ are identified by PCR from DNA extracted from various K5-Cre-ER T(tg/0> /RXR ⁇ L2/+ mouse organs, twelve weeks after the first Tamoxifen treatment.
  • Figure ID The obtaining of L ⁇ RXR ⁇ alleles mediated by K14-Cre-ER T2 induced by Tamoxifen in the epidermis of adult mice is illustrated by PCR analysis of the genomic DNA extract from the epidermis (E) and from the dermis (D) isolated two weeks after the first injection of Tamoxifen (0.1 mg) (+) or of the vehicle (oil) (-) .
  • the genotype of the mice is indicated and the PCR fragments corresponding to the RXR ⁇ (+ ), L2 and L- alleles are indicated.
  • Figure 2 Abnormalities generated by the inactivation of RXR ⁇ mediated by K5-Cre-ER T and K14-Cre-ER T2 induced by Tamoxifen, in the skin of adult mice.
  • FIG. 2A Ventral view of a "mutant" (mt) female mouse
  • Figure 2B Dorsal view of the same animals.
  • mutant female mouse K5-Cre-ER ⁇ ltg/0) /RXR ⁇ 2/ ⁇ , with the arrow indicating one of the cysts visible under the surface of the skin.
  • Figure 2D Dorsal view of the "mutant" (mt) female mouse K5-Cre-ER (tg/0) /RXR ⁇ L2/" / twenty-eight weeks after the start of Tamoxifen treatment.
  • the arrow indicates a minor skin lesion.
  • FIGS 2E-H Histological analysis. Histological sections 2 ⁇ m thick of the ventral skin of "control" (E and G) and “mutant” (F and H) mice, sixteen weeks after the start of the treatment. Hair follicles (hf) , utriculi (u) and dermal cysts (dc) are indicated. The arrow tips in (H) indicate the Langerhan's cells, whose number is increased several fold in the epidermis of the mutant mice. Increased cellularity can be noted in
  • FIG. 21 Immunohistochemistry of keratin 6 (K6) on sections of skin of "control" mice (I) and of “mutant” mice ( ) , sixteen weeks after the first Tamoxifen treatment.
  • the red color corresponds to the labeling with the antibody directed against K6, and the Cyan color corresponds to staining with DAPI .
  • K6 is normally expressed in the outer root sheath of the hair follicle (hf) but not in the epidermis (I) and is abnormally expressed in the hyperproliferative epidermis of "mutant" mice (J) .
  • Scale (in I) I 25 ⁇ m.
  • the arrows in E to J indicate the dermis-epidermis junction.
  • Figures 2K, L Appearance of the skin of a mutant female mouse K14-Cre-ER T2(tg/0) /RXR ⁇ L2/ 2 •
  • K magnification on the ventral region, sixteen weeks after the start of Tamoxifen treatment (0.1 mg per injection); the white arrow indicates a cyst and the black arrow indicates a utriculus containing melanosomes.
  • L dorsal view of the same mutant. The arrow indicates one of the skin lesions associated with a hairless region.
  • Figure 3 Similarities and differences between the skin abnormalities present in a double "mutant" mouse K5-Cre-ER ⁇ (tg/0) /RXR ⁇ L2/L2 /RXRp _/" induced with Tamoxifen and a "null" VDR mouse.
  • FIGS 3A, E K5-Cre-ER T(tg/0) /RXR ⁇ L2/L2 /RXR ⁇ _/ ⁇ mouse eighteen weeks after the first treatment with Tamoxifen (1 mg of Tamoxifen per injection) (A) and fourteen-week old VDR "/_ mouse (E) .
  • the arrows in (A) indicate the skin lesions.
  • (B, C) and (F, G) histological analysis of 2 ⁇ m thick sections of dorsal skin of the animals presented in (A) and (E) respectively.
  • FIGS 3D, H Immunohistochemistry of keratin 6 (K6) on mouse skin sections removed from the animals presented in (A) and (E) respectively.
  • K6 is expressed in the utriculus, but not in the epidermis of the skin of the VDR ⁇ /_ mice, whereas K6 is expressed in the entire hyperproliferative epidermis of the "double mutant" mice RXR ⁇ /RXR p (D) .
  • Utriculus (U) dermal cyst (dc) .
  • the arrows in (B-H) indicate the dermis-epidermis . junction.
  • Figure 4 Selected targeted inactivation of the SNF2 ⁇ gene in the epidermis of adult mice.
  • Figure 4A Schematic representation of the wild-type SNF2 ⁇ alleles (+) , L3, L2+ and L- .
  • the size of the DNA segments revealed by the 5' probe, after enzymatic digestion of the genomic DNA by BamHI is indicated.
  • the L3 allele in the ES cells was obtained by homologous recombination using a strategy similar to that described for the F9 cells in Sumi-Chinose et al . , 1997.
  • mice were then crossed with RXR ⁇ + " mice (Kastner et al . f 1994) so as to produce aP2-Cre-ER ⁇ 2(tg/0) /RXR ⁇ L2/" mice.
  • Such four week old mice were treated (+) or not (-) with Tamoxifen (1 g/day) for five days, and the adipose tissue collected one month after the last injection of Tamoxifen.
  • the DNA was extracted from the adipose tissue, or after separation of the adipocytes (80% purified adipocytes) from the connective tissue and blood vessels (nonadipocytes) .
  • FIG. 6 Phenotypic analysis of mice after conditional somatic mutagenesis of RXR ⁇ in the adipocytes.
  • the weight of the control mice aP2-Cre-ER T tg/0) RXR « i2/+ (CT) and aP2-Cre-ER T2(tg/0, /RXR ⁇ L2/ ⁇ (mutants; KO) was determined once per week.
  • each group of animals was composed of 10 to 15 males. The animals were fed either with the normal food (AN) or food enriched with fat and with glucose (AR) .
  • C 10 ⁇ m cryosections of subcutaneous adipose tissue of 6-month old CT (a and c) and KO (b and d) mice, fed with AN (a and b) or AR (c and d) .
  • Scale 160 ⁇ m.
  • the levels of triglycerides (D) , glucose (E) and insulin (F) were determined on the serum of 4- to 5-month old CT and KO animals, fed with AN or AR.
  • the glucose assay was carried out after starving the animals for 12 hours, * p ⁇ 0.05.
  • FIG. 7 Selective targeted inactivation of the RXR « gene in the murine hepatocytes .
  • ⁇ AT-Cre-ER ⁇ (tg/0> mice which express Cre-ER T under the control of the promoter of the ⁇ -1-antitrypsin ( ⁇ AT) gene in about 50% of the hepatocytes (Ima ⁇ et al . , 2000) were crossed with RXR ⁇ L2/L2 mice so as to produce ⁇ AT-Cre-ER T(tg/0) /RXR ⁇ L2 2 mice.
  • Such three month old mice were treated with Tamoxifen (1 g/day) for five days, and the heart and liver removed seven days after the first injection of Tamoxifen (day 7), from one and three animals respectively.
  • the heart and the liver were also removed from one and three animals respectively of the same genotype without treatment with Tamoxifen (Day 0) .
  • the DNA was extracted from these tissues, and after digestion with BamHI, the alleles of RXR ⁇ were analyzed by Southern blotting. An autoradiogram is presented. No excision is observed in the heart at day 0 or 7, or in the liver at day 0. Conversely, excision is observed in the liver of mice seven days after the treatment with Tamoxifen; this excision is materialized by the appearance of a band at 8.5 kb on the autoradiogram, corresponding to the L ⁇ allele.
  • Figure 8 Site-specific recombination of RXR ⁇ in the liver.
  • Lanes 15-17 correspond to the DNA isolated from the livers of animals 7 days after partial hepatectomy
  • Figure 10 Nuclear translocation of Cre-ER T2 and Cre-ER X3 following a two-day treatment at various OHT doses .
  • the excision levels induced by 1 and 0.1 mg of OHT are similar for the two lines; on the other hand, the excision is more efficient in the K5-Cre-ER 3 than in the K5-Cre-ER T2 mice at the doses of 0.01 and 0.001 mg of OHT.
  • Figure 12 Rate of papillo a formation in RXR ⁇ e ⁇ mice.
  • A Timing of Tarn-induced RXR ⁇ ablation in epidermal keratinocytes, and DMBA/TPA tumorigenesis .
  • Tarn- treatment (0.1 mg for 5 consecutive days) was performed either 16 days before (bar a) or 7 weeks after (bar b) topical DMBA application.
  • TPA was topically applied twice a week (arrows) for up to 30 weeks.
  • B Papillomas in DMBA/TPA-treated RXR ⁇ ep ⁇ / ⁇ mice.
  • the number of papillomas induced by the DMBA/TPA treatment was determined macroscopically in 6 CT and 6 RXR ⁇ ep ⁇ _ mice, and plotted versus the number of weeks after the start of the carcinogenic treatment. Values are expressed as mean +/- SEM.
  • C Dorsal view of CT (K14-Cre- ER ⁇ 2 ⁇ 0 /0 ) /R ⁇ R ⁇ L2/2 ⁇ (left) and R ⁇ R ⁇ e -/ ⁇ ( right ) mice 25 weeks after the start of the DMBA/TPA treatment.
  • D Length distribution of papillomas in CT and RXR ⁇ ep mice. The number of tumors of a given length was determined on 6 CT and 6 RXR ⁇ e " ⁇ ⁇ mice, 30 weeks after the start of the DMBA/TPA treatment. Values are expressed as mean +/- SEM.
  • Figure 13 Histological analysis of skin tumors induced by DMBA/TPA treatment.
  • A Representative hematoxylin and eosin stained 5 ⁇ m- thick paraffin sections from CT biopsies taken 25 (a - b) and 30 (c) weeks after the start of the DMBA/TPA treatment.
  • B Representative hematoxylin and eosin stained 5 ⁇ m-thick paraffin sections from RXR ⁇ ep biopsies taken 25 (a-c) and 30 (d-i) weeks after the start of the DMBA/TPA treatment.
  • B. Number of melanocytic growths in skin of CT and RXR ⁇ ep ⁇ /_ mice 30 weeks after DMBA and DMBA/TPA treatments, as indicated. Values are expressed as mean +/- SEM (n 6) . *, p ⁇ 0.05; ***, p ⁇ 0.001.
  • Figure 15 Malignant melanomas in lymph nodes of DMBA- treated RXR ⁇ ep_ ⁇ mice.
  • the mouse lines RXR ⁇ +/" , VDR _/" and K5-Cre-ER T have been previously described in Yoshizawa et al . (1997), Kastner et al . (1994) and Indra et al . (1999).
  • the transgene K14-Cre-ER T2 was constructed by replacing the K5 promoter region of the vector pK5-Cre-ER T2 (Indra et al . , 1999) with the Sail DNA fragment of the promoter/enhancer region of 2 kb of the K14 human keratin gene, isolated from Phr2 (gift from S. Werner) .
  • the transgenic mice were generated in accordance with the article by Indra et al . (1999) .
  • the transgene aP2-Cre-ER T2 was constructed as follows: a 5.4 kb fragment containing the aP2 promoter was amplified by PCR from mouse genomic DNA with the aid of the LA-PCR kit (Perkin-Elmer, New Jersey) , with the oligonucleotides
  • the genomic DNA is isolated from tissues according to the protocol described in the article by Indra et al . (1999) .
  • the epidermis is separated from the dermis after treating the skin of the tail with the enzyme dispase (4 mg/ml in PBS, GIBCO-BRL) for 1 to 2 hours at room temperature.
  • the genotyping of the RXR ⁇ cells is carried out by PCR (polymerase chain reaction) using the primers ZO 243 (5'-TCC TTC ACC AAG CAC ATC TG-3') (SEQ ID No. 9) (located in exon 3) and ZO 244 (5'-TGC AGC CCT CAC AAC TGT AT-3' ) (SEQ ID No.
  • the primers ZO 243 and UD 196 (5'-CAA CCT GCA CTT GTC ACT TAG-3' ) (SEQ ID No. 11) (located in the intron between exons 4 and 5) were used in the polymerase chain reaction to reveal the L " allele; this amplification reaction generates a fragment of 400 bp.
  • the primers ZO 243 and RU 178 (5' -ATG TTT CAT AGT TGG ATA TC-3' ) (SEQ ID No. 12) (located in the neo cassette) are used in the polymerase chain reaction to reveal the (-) allele; this PCR reaction generates a fragment of 500 bp.
  • the genomic DNA is digested with BamHI and the probe used is the probe X4 (3 kb BamHI-Xbal fragment of the RXR ⁇ gene) (Metzger et al . , 1995).
  • Tamoxifen (Sigma) solutions are prepared according to the protocol described by Metzger and Chambon (2001) .
  • 1 mg of Tamoxifen dissolved in 100 ⁇ l of sunflower oil is intraperitoneally injected into a transgenic mouse K5-Cre-ER T for five consecutive days, and then again for three consecutive days, two, four and six weeks later.
  • the K14-Cre-ER T2 transgenic mice are intraperitoneally injected with 0.1 mg of Tarn dissolved in 100 ⁇ l of sunflower oil for five consecutive days, while the ⁇ P2-Cre-ER ⁇ 2 and ⁇ AT-Cre-ER ⁇ mice are treated with 1 mg of Tarn.
  • the skin biopsies from animals of the same age and of the same sex were prepared at the level of the same sites of the body.
  • the skin samples are fixed in glutaraldehyde
  • Vectashield medium (Vector Laboratories) containing
  • DAPI 6-diamidino-2-phenylindole dihydrochloride
  • Tumors (8 - 16 mm) were excised 22 weeks after DMBA application and immediately embedded in OCT and frozen on dry ice. 10 ⁇ m-thick frozen sections from CT and RXR ⁇ ep tumors were reacted with primary antibodies [mouse monoclonal anti-KlO, rabbit polyclonal anti-K5 (gifts from Prof. Brigitte Lane, Cell Structure research group, University of Dundee) , rabbit polyclonal anti-Kl (Babco) , rabbit polyclonal anti-K13 (gift from Prof. S.
  • adipose tissue samples are removed from animals perfused with PFA, fixed with the aid of formaldehyde (20% in PBS) and then frozen in OCT (Tissue-Tek compound, Sakura) . Cryosections of 10 ⁇ m are stained with hematoxylin and eosin.
  • the liver is removed from animals, rinsed in PBS, fixed in a Boin solution and then embedded in paraffin. Sections of 6 ⁇ m are stained with hematoxylin and eosin.
  • Tumors from 25 animals were excised at 25 or 30 weeks after DMBA application, fixed in Bouin' s fixative and embedded in paraffin. 5 ⁇ m sections were stained with hematoxylin and eosin. Electron microscopy was performed as described (Li et al . , 2001).
  • Subiliac lymph nodes were isolated 30 weeks after DMBA application, fixed in 4 % paraformaldehyde, photographed and embedded in paraffin. 5 ⁇ m-thick sections were stained with hematoxylin and eosin.
  • the assay of the triglycerides and of cholesterol is carried out according to Peters et al .
  • CT and RXR ⁇ ep mice were shaved 7 and 2 days before DMBA treatment, and every second week for 30 and 15 weeks, respectively. 8 weeks after DMBA treatment, all-trans retinoic acid (t-RA, 20 n oles in ethanol) or vehicle was topically applied 15 min before each TPA application.
  • the length of the papillomas and the diameter of the melanocytic were measured with a Vernier calliper on isofluorane anesthesised mice.
  • hair loss was observed six to seven weeks after the first treatment with Tamoxifen in the ventral region of the mice; this was not observed in the mice treated with oil alone without Tamoxifen, or in the "control" mice of the same litter treated with Tamoxifen (K5-Cre-ER T(tg/0) /RXR ⁇ L2/+ ) .
  • Cre-ER T2 whose activity may be induced by a gentler treatment with Tamoxifen (0.1 mg for five days) (Indra et al . , 1999) .
  • K14-Cre-ER T2(tg/0) /RXR ⁇ L2/L2 mice were treated simultaneously with "control", K14-Cre-ER ⁇ 2
  • RXR ⁇ L ⁇ allele was detected in the epidermis of K14-Cre-ER ⁇ (tg/0) /RXR ⁇ 2/ 2 mice, indicating that RXR ⁇ was excised in the majority, if not all, of the stem cells of the epidermis.
  • the inactivation of RXR ⁇ also appears in the other epithelia of other organs in which the K14 promoter is active (Wang et al . , 1997) (that is to say the tongue, esophagus, stomach) .
  • the underlying dermal and epidermal histological abnormalities are also similar to those observed above for the K5-Cre-ER T(tg/01 /RXR ⁇ L2/L2 mice treated with Tamoxifen.
  • these "constitutive" and epidermis-specific RXR ⁇ mutants develop progressive alopecia with typical characteristics of degenerated hair follicles, utriculi and dermal cysts, which may all be attributed to defects in the hair cycle. Furthermore, these mutants also exhibit interfollicular hyperproliferation of the keratinocytes, as well as abnormal terminal differentiation (with expression of K6) , and an increase in the dermal cellularity associated with an inflammatory reaction of the skin.
  • RXRp is expressed in the epidermis of mice, the skin of adult RXRp _/ ⁇ mutants appears normal
  • Histology of the hairless skin shows disappearance of the hair follicles and the presence of utriculi and of dermal cysts (Figure 3B) .
  • the epidermis is highly hyperplastic and hyperkeratinized (compare Figures 3B and 3C with Figures 2E and 2G, and Figures 2F and 2H) .
  • Abnormal expression of K6 is observed through the whole epidermis ( Figure 3D) and an inflammatory reaction with an increase in cellularity is also observed ( Figure 3B) .
  • the epidermis is covered with a crust and is more hyperplastic and hyperkeratinized.
  • K5-Cre-ER ⁇ (tg/0) /RXR ⁇ L2/ 2 /RXR ⁇ "/ VRXR ⁇ "/” do not reveal an additional role of RXR ⁇ in the adult skin.
  • RXRp may partially compensate for the loss of RXR ⁇ function.
  • the functional redundance is more pronounced in the males than in the females, since the male and female double mutants RXR ⁇ /RXR ⁇ are affected in a similar manner unlike the single mutants.
  • VDR "knock-out” mice mice in which the two alleles of the VDR gene are inactive (VDR "knock-out” mice) develop progressive secondary alopecia, suggesting that VDR is involved in the hair cycle rather than in primary hair growth (Yoshizawa et al . , 1997; Li et al . , 1997; Li et al . , 1998).
  • the inventors constructed a mouse carrying floxed SNF2 ⁇ alleles (L2+; Figure 4A) and used the transgenic mouse line K14-Cre-ER T2 in which Tamoxifen effectively induces Cre-mediated recombination in the keratinocytes of the basal layer of the epidermis.
  • Eight-week-old K14-Cre-ER ⁇ 2(tg/0) /SNF2 ⁇ L2 L2 and K14-Cre-ER ⁇ 2(0 0) /SNF2 ⁇ L2/L2 mice were treated with Tamoxifen for five days at the rate of 0.1 mg/day or with oil (-) .
  • Tamoxifen induces the exicision of the floxed fragment of the SNF2 ⁇ gene only in the epidermis, because the K14 promoter is active only in this tissue and not in the dermis.
  • Tamoxifen induces no excision in K14-Cre-ER ⁇ 2(0/0> /SNF2 ⁇ 2/L2 "control" mice whose cells do not contain a Cre-ER T2 transgene.
  • mice To carry out the spatiotemporally controlled site-specific mutagenesis in the adipocytes, the inventors created transgenic mice called aP2-Cre-ER T2 expressing the Cre-ER T2 fusion protein under the control of the promoter of the gene encoding adipose protein 2 (aP2) which is specifically active in the adipocytes (Ross et al . , 1990) .
  • aP2(tg/0) mice were first crossed with RXR ⁇ 2/+ mice so as to produce aP2-Cre-ER ⁇ 2(tg/0, /RXR ⁇ 2/ 2 mice.
  • mice were then crossed with RXR ⁇ +/ ⁇ mice (Kastner et al . , 1994) so as to produce aP2-Cre-ER T2(tg/0) /RXR ⁇ L2/ ⁇ mice.
  • RXR ⁇ +/ ⁇ mice Kastner et al . , 1994
  • Such four week old mice were treated (+) or not (-) with Tamoxifen
  • the DNA was extracted from the adipose tissue, or after separation of the adipocytes (80%- purified adipocytes) from the connective tissue and blood vessels (non-adipocytes) .
  • the DNA is then digested with BamHI and then separated by agarose gel electrophoresis, transferred onto nylon membranes and then hybridized with the radiolabeled X4 probe.
  • the corresponding radiographs are presented in Figure 5.
  • mice treated with Tamoxifen exhibit excision in the RXR ⁇ gene in about 50% of the liver cells, which indeed corresponds to the expression, in the form of a mosaic, of the Cre-ER T protein in the hepatocytes of the oAT-Cre-ER T mouse.
  • Cre-ER T2 V400 with G (Cre-ER mutant M543A/L544A, called "Cre-ER T3 ").
  • Cre-ER T3 G
  • Transgenic mice expressing Cre-ER T3 under the control of the K5-promoter were obtained, and a line expressing the chimeric recombinase at similar levels to those detected in the K5-Cre-ER T and K5-Cre-ER T2 lines was generated (see Figure 9, and results not presented) .
  • the sensitivity to OHT was tested in a first instance by analyzing the intracellular location of the chimeric proteins. Whereas after treatment with 0.1 mg of OHT, Cre-ER T2 and Cre-ER T3 are both located in the cellular nuclei, Cre-ER T3 is present in a larger fraction thereof at lower doses of Tarn or OHT. At the dose of 0.001 mg, about 1/3 of the nuclei are strongly labeled with anti-Cre antibodies in the basal layer of the epidermis of K5-Cre-ER T3 mice, whereas no positive nucleus is observed in the skin of K5-Cre-ER T2 mice ( Figure 10) .
  • DMBA 12-O-tetradecanoylphorbol 13- acetate
  • TPA 12-O-tetradecanoylphorbol 13- acetate
  • CT control mice which carried, in epidermal keratinocytes, one WT (+) and one RXR ⁇ L- allele, and two RXR ⁇ L2 alleles, respectively.
  • CT and RXR ⁇ ep female mice were topically-treated 16 days after the first Tarn injection with a single dose of DMBA (50 ⁇ g) , and then twice a week with TPA (5 ⁇ g) for 25-30 weeks
  • FIG. 12A Seven to eight weeks after DMBA application, small papilloma were observed in all CT and RXR ⁇ ep mice, and their number and size increased with time (Fig.l2B and data not shown). Interestingly, RXR ⁇ ep mice developed approximately twice as many tumors as CT mice, and 30 weeks after the start of
  • ⁇ 50 tumors from 6 CT and 6 RXR ⁇ ep mice were histologically examined.
  • almost all tumors analysed 25 weeks after the start of DMBA/TPA treatment of CT mice were benign papillomas characterised by skin folds integrated by a core of connective tissue and lined by an acanthotic, hyperkeratotic, stratified squamous epithelium (Fig. 13Aa and 13C (table)).
  • 35 % of the papillomas exhibited an atypical hyperplasia, 5% displayed an in si tu carcinoma, but no focal carcinoma could be detected (Fig. 13Ab and 13C (table), and data not shown) .

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Abstract

L'invention concerne un organisme métazoaire, à l'exception de l'homme, et en particulier une souris, caractérisée en ce qu'au moins une cellule de cet organisme comprend au moins une protéine de fusion entre une recombinase Cre et un domaine de liaison de ligand modifié du récepteur alpha d'oestrogène du noyau, permettant une induction de la recombinase fusionnée inactive par des oestrogènes synthétiques, mais pas par des oestrogènes naturels, et en ce qu'une ou plusieurs séquences d'ADN d'intérêt, appartenant au génome de cet organisme, dans laquelle est insérée un ou plusieurs sites de reconnaissance de cette protéine recombinase. L'invention concerne aussi les procédés utilisant cet organisme pour le criblage de médicaments, la mutagenèse et l'analyse de la fonction biologique de la ou des séquences d'ADN d'intérêt, en particulier de gène(s) d'intérêt, tel que RXRα.
PCT/IB2001/002246 2000-10-03 2001-09-28 Sousris transgenique pour recombinaison ciblee a mediation par cre-er modifie WO2002028175A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
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WO2005040212A2 (fr) * 2003-10-24 2005-05-06 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Commutateurs geniques orthogonaux
WO2006047367A2 (fr) * 2004-10-22 2006-05-04 Therapeutic Human Polyclonals, Inc. Suppression de l'expression d'immunoglobulines endogenes
CN100387722C (zh) * 2004-02-18 2008-05-14 中国人民解放军军事医学科学院生物工程研究所 一种中枢神经系统特异性表达Cre重组酶的转基因小鼠的制备方法
EP2067858A1 (fr) 2007-12-07 2009-06-10 Universidad de Sevilla Modèles d'animaux pour maladies neurodégénératives
WO2009138544A1 (fr) 2008-05-16 2009-11-19 Proyecto De Biomedicina Cima, S.L. Adénovirus auxiliaires auto-inactivants pour la production d'adénovirus de recombinaison de capacité élevée
WO2018129203A3 (fr) * 2017-01-06 2018-08-16 The Regents Of The University Of California Procédé d'administration temporelle de médicament spécifique au tissu et recombinaison induite d'acide nucléique
CN112921052A (zh) * 2019-12-06 2021-06-08 中国科学院分子细胞科学卓越创新中心 体内细胞增殖标记与示踪系统及其应用

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WO2005040212A2 (fr) * 2003-10-24 2005-05-06 Istituto Di Ricerche Di Biologia Molecolare P Angeletti Spa Commutateurs geniques orthogonaux
WO2005040212A3 (fr) * 2003-10-24 2005-07-28 Angeletti P Ist Richerche Bio Commutateurs geniques orthogonaux
CN100387722C (zh) * 2004-02-18 2008-05-14 中国人民解放军军事医学科学院生物工程研究所 一种中枢神经系统特异性表达Cre重组酶的转基因小鼠的制备方法
WO2006047367A2 (fr) * 2004-10-22 2006-05-04 Therapeutic Human Polyclonals, Inc. Suppression de l'expression d'immunoglobulines endogenes
WO2006047367A3 (fr) * 2004-10-22 2006-06-15 Therapeutic Human Polyclonals Suppression de l'expression d'immunoglobulines endogenes
EP2067858A1 (fr) 2007-12-07 2009-06-10 Universidad de Sevilla Modèles d'animaux pour maladies neurodégénératives
WO2009138544A1 (fr) 2008-05-16 2009-11-19 Proyecto De Biomedicina Cima, S.L. Adénovirus auxiliaires auto-inactivants pour la production d'adénovirus de recombinaison de capacité élevée
WO2018129203A3 (fr) * 2017-01-06 2018-08-16 The Regents Of The University Of California Procédé d'administration temporelle de médicament spécifique au tissu et recombinaison induite d'acide nucléique
CN112921052A (zh) * 2019-12-06 2021-06-08 中国科学院分子细胞科学卓越创新中心 体内细胞增殖标记与示踪系统及其应用
CN112921052B (zh) * 2019-12-06 2023-07-21 中国科学院分子细胞科学卓越创新中心 体内细胞增殖标记与示踪系统及其应用

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